Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

11

Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

Abstract

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressantactions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

Ketamine is metabolised in vivo via P450 enzymatic transformations. (i) (R,S)-Ketamine (KET) is selectively demethylated to give (R,S)-norketamine (norKET). (ii) NorKET can be then dehydrogenated to give (R,S)-dehydronorketamine (DHNK). (iii) Alternatively, norKET can be hydroxylated to give the hydroxynorketamines (HNKs). (iv) (R,S)-KET can also be hydroxylated at the 6- position to give either the E-6-hydroxyketamine ((2S,6R;2R,6S)-HK)) or Z-6-hydroxyketamine ((2S,6S;2R,6R)-HK)). (v) Demethylation of (2S,6R;2R,6S)-HK yields the production of (2S,6R;2R,6S)-hydroxynorketamine (HNK). (vi) Demethylation of (2S,6S;2R,6R)-HK further gives (2S,6S;2R,6R)-hydroxynorketamine (HNK).

a, Chronic social defeat stress and social interaction/avoidance test timeline. (b-c), Administration of (R,S)-ketamine (KET) or MK-801did not affect b, locomotor activity or c, total number of compartmental crosses in the social interaction apparatus. Data are means ± S.E.M. ***p<0.001. SAL, saline (statistical analyses and n numbers see ).

Acute and sustained antidepressant and anti-anhedonic effects of (2R,6R)- and (2S,6S)-hydroxynorketamine

a, A single injection of (2R,6R)-HNK resulted in dose-dependent antidepressant-like responses in the learned helplessness test at the doses of 5-75 mg/kg. b, A single injection of (2S,6S)-hydroxynorketamine (HNK) induced antidepressant-like effects in the learned helplessness test at the dose of 75 mg/kg. c, Administration of (2R,6R)-HNK induced dose-dependent antidepressant effects in the 1- and 24-hour forced-swim test. d, Administration of (2S,6S)-HNK at the dose of 25 mg/kg induced antidepressant effects in the 1- and 24-hour forced-swim test. e, Despite the greater antidepressant efficacy of (2R,6R)-HNK, administration of (2S,6S)-HNK (HNK) results in higher brain hydroxynorketamine levels compared to (2R,6R)-HNK. f, (2R,6R)-HNK manifested dose-dependent antidepressant-like effects in the novelty-suppressed feeding test. g, Similar to (R,S)-ketamine (KET), the antidepressant-like effects of (2R,6R)-HNK in the forced-swim test persisted for at least 3 days post-treatment. h, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decreases in sucrose preference. i, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decrease in female urine sniffing preference, specifically in mice that developed an anhedonic phenotype. Administration of (2R,6R)-HNK was not associated with changes in j, locomotor activity or k, total compartmental crosses in the social interaction test following chronic social defeat stress. Data are means ± S.E.M. *p<0.05, **p<0.01, ***p<0.001. SAL, saline (statistical analyses and n numbers see ).

Effects of (2R,6R)-hydroxynorketamine on synaptoneurosome protein and protein phosphorylation levels

A single administration of (R,S)-ketamine (KET, 10 mg/kg) or (2R,6R)-hydroxynorketamine (HNK, 10 mg/kg) (a,b), did not alter levels of mTOR or phosphorylated mTOR 1- or 24-hours post-injection in the hippocampus of mice. (b-i), Administration of (R,S)-KET or (2R,6R)-HNK did not alter levels of (c,d), mTOR/phosphorylated mTOR, (e,f), eEF2/phosphorylated eEF2, (g,h), proBDNF/mBDNF, or (i,j), GluA1/GluA2 in the prefrontal cortex of mice. The values for the phosphorylated forms of proteins were normalised to phosphorylation-independent levels of the same protein. Phosphorylation-independent levels of proteins were normalised to GAPDH. Data are means ± S.E.M, and was normalised to the saline-treated control group for each protein. Images are cropped; see for complete blot images *p<0.05 (statistical analyses and n numbers see ). Abbreviations: eEF2, eukaryotic translation elongation factor 2; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; mBDNF, mature brain-derived neurotrophic factor; mTOR, mammalian target of rapamycin; proBDNF, pro-brain-derived neurotrophic factor; SAL, saline.

a, Startle amplitude as measured in the pre-pulse inhibition task was not affected by administration of (R,S)-ketamine (KET) or (2R,6R)-hydroxynorketamine (HNK). (b,c), Response rate of overall lever pressing per sec in the drug discrimination paradigm was not changed by administration of b, (R,S)-KET, (2R,6R)-HNK or c, phencyclidine (PCP). d, Unlike ketamine, (2R,6R)-HNK did not alter drug intake in the self-administration task in mice. Data are means ± S.E.M. *p<0.05 (statistical analyses and n numbers see ). SAL, saline.